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Louise McClelland
13th October 2010
Filaggrin
 Polyprotein profilaggrin
 Epidermal differentiation
 cDNA library
 Somatic hybrids
 M13 sequencing
 Long range PCR
 Ichthyosis vulgaris
 Atopic dermatitis (eczema)
 Asthma
 Ancient mutations

 Filaggrin

protein
Processing and function
 Gene
ID and characterisation
 Mutation analysis
 Filaggrin
is synthesised as a giant precursor
protein, profilaggrin
 Filament-aggregating protein
 Interact with keratin intermediate filaments
in terminally differentiating mammalian
epidermis
 Very important in facilitating epidermal
differentiation and maintaining skin barrier
function
 Implicated in a no. of keratinizing disorders
 1981

- Term Filaggrin (FLG) coined
Described a class of structural proteins that have
been isolated from the stratum corneum
 1984-1985
– Filaggrin and profilaggrin protein
characterisation
 1989 – FLG gene cloned and mapped
 1992-1993 Gene characterisation
 2006 – LOF mutations ID’d in ichthyosis
vulgaris and atopic dermatitis
 2007 – Further mutation analysis
1980
1990
2000
2010
 >400
kDa
 N-terminal domain


2 subdomains
?role in regulation of profilaggrin by Ca2+
 Repeat


region
10-12 tandemly arranged filaggrin repeats
Linker regions between repeats
 C-terminal
domain
1984-1985
1980
1990
2000
2010
 During
terminal differentiation of granular
cells profilaggrin is proteolytically cleaved
into;



Multiple 37 kDa (342 AA) filaggrin peptides
N and C domains
Linker regions between repeats
 Phosphorylation
of profilaggrin believed to
prevent premature cleavage
Aggregates to keratin intermediate filaments in
cytoskeleton
 Creates a dense insoluble protein-lipid keratin
matrix cross linked by transgluaminases
 Causes collapse of granular cells into flattened
anuclear squames
 Scaffold for the attachment of cornified cell
envelope (CCE) proteins and lipids
 CCE

Outermost barrier layer of skin
 Prevents water loss
 Impedes allergens, toxic chemicals and infectious
agents

 Post
translational conversion of arginine to
citrulline (Deimination/citrulination)
 Promotes unfolding and degradation into
hygroscopic AA and derivatives
 Contributes to Natural moisturising factor
(NMF)

NMF responsible for maintaining hydration in low
environment humidity
 Differences
between mouse and human FLG
complicated ID


Anti-mouse FLG antibodies do not recognise
human FLG
cDNA clones encoding mouse FLG do not
recognise human FLG
 cDNA
library of human foreskin epidermal
tissue created in a λ phage
 Screened library using human anti-filaggrin
monoclonal antibody
 Antibody positive clones purified from
plaques (12 clones)
1989
1980
1990
2000
2010
Griffiths, 1999
 cDNA
inserted into M13 vectors
Strachan and Read, 2010
 Longest



clone contained ORF of 416 AA
Beginning and end share homology
Showed encodes a polyprotein precursor of
filaggrin
Identified linker sequences are removed to
create monomers
 Somatic
cell hybrids
 Recorded what human
chromosomes retained
in each hybrid
 Examined hybrids DNA
by Southern blot
 Filaggrin sequence
probe
 Only hybrids retaining
chromosome 1
Griffiths, 1999
 RNA
probes for in situ
hybridisation
 Synthesised from earlier
plaques (in pGEM-3B
vector)
 Refined chromosomal
position to 1q
 Cloned
profilaggrin cDNA was compared to
genomic profilaggrin to identify the
exon/intron boundaries

Contains 3 exons
1992-1993
1980
1990
2000
2010
Dry, flaky skin
 First
investigated in ichthyosis
vulgaris (IV)



Most common and mildest ichthyosis
Poorly formed stratum corneum
prone to water loss
Predisposition to eczema and
associated asthma
Keratosis pilaris
Smith et al., 2006
1980
Palmar hyperlinearity of
palms and soles
1990
2006
2000
2010
 Absence
of filaggrin in keratinocytes of IV
patients by immunoblotting
 Decreased FLG mRNA
 Recessive mouse model flaky tail (ft)


Histologically similar to IV
Strong genetic linkage to mouse FLG locus
 IV
linkage to 1q21 in an American family
 15

kindred examined
Irish, Scottish and Americans of European decent
 Long


range PCR of FLG
R501X
2284del4
Allele F = ~4% in European ancestry
 Both
cause PTC in 1st filaggrin repeat
 Semi dominant inheritance


Heterozygotes = mild phenotype
Homozygotes = severe phenotype
 Noted
several patients had atopic dermatitis
and a few had asthma
 Atopic
dermatitis (eczema)
 Allergies
 Asthma
 FLG mutations suspected in atopic disease
because of link between IV and eczema
 Identified
R501X and 2284del4 mutations in
atopic disease
 Significant association between these
mutations and;


Atopic dermatitis
Asthma in the context of atopic dermatitis
 Carried
by 9% of people of European origin
Palmer et al., 2006
1980
2006
1990
2000
2010

Several mutations now identified


Other populations with FLG mutations identified


Japanese, Mexican, Basque, Indian
Distinct haplotypes associated with some mutations




All common mutations null mutants
R501X, 2284del4, R2447X, S3247X
R501X and 2284del4 absent from non-Europeans
Ancient mutations
Similar pathogenicity of 3’ mutations as 5’

Immunoblotting showed still greatly reduced filaggrin
2007
Sandilands et al., 2007
1980
1990
2000
2010




Major predisposing gene for atopic disease
Complex disease model emerging
Refocused attention on skin barrier rather than immunity
in eczema research
FLG mutations may be modifying factors in other
conditions



E.g. X-linked ichthyosis
Reduced no. of FLG repeats may predispose to dry skin
Hypothesis of why mutations so common in Europe
(particularly Ireland)

Mutation advantage at some point in history


Allows a degree of natural vaccination against antigens e.g. plague
Identifying when the mutations arose may help explain
why so prevalent







Brown, Genome, BIOS, 1st edition, 1999
Griffiths et al., Modern Genetic Analysis, Freeman
and Co. NY, 1st edition, 1999
McKinley-Grant et al, PNAS 86: 4848-4852; 1989
Palmer et al., Nat Genet 38: 441-446; 2006
Presland et al., J Biol Chem 267: 23772-23781; 1992
Sandilands et al., Nat Genet 39: 650-654; 2007
Sandilands et al., J Cell Sci 122: 1285-1294; 2009




Good review
Smith et al., Nat Genet 38: 337; 2006
Steinert et al., PNAS 78: 4097-4101; 1981
Strachan and Read, Human Molecular Genetics,
Garland Science, 4th edition, 2010